Mating system parameters in species of genus Prosopis (Leguminosae).

doi:10.1111/J.1601-5223.2000.00019.X

Hereditas 132: 19-27

(2000)

Mating system parameters in species

of

genus

Prosopis

(Leguminosae)

CECILIA BESSEGA*,

LAURA

FERREYRA, NORMA

JULIO?,

SOLEDAD

MONTOYA,

BEATRIZ

SAIDMANT

and

JUAN

C.

VILARDIT

Departamento de Ciencias Biologicas, Facultad Ciencias Exactas

y

Naturales, Universidad de Buenos Aires,

Buenos Aires, Argentina

Bessega, C., Ferreyra,

L.,

Julio,

N.,

Montoya,

S.,

Saidman, 9. and

Vilardi,

J.

C.

2000.

Mating system parameters in species

of genus

Prosopis

(Leguminosae).-Hereditas

132:

19-27. Lund, Sweden. ISSN 0018-0661. Received October

21,

1999.

Accepted January 10,

2000

The section Algarobia of genus

Prosopis

involves important natural resources in arid and semiarid regions of the world.

Their rationale use requires a better knowledge of their biology, genetics and mating system. There are contradictory

information about their mating system. Some authors claim they are protogynous and obligate outcrosser. However, some

evidence have been shown indicating that they might not be protogynous and that they might be somewhat self-fertile.

The current paper analyses genetic structure and mating system parameters in populations of seven species of this section

from South and North America based on isozyme data. In all species a significant homozygote excess was found in the

offspring population but not in mother plant genotypes. Multilocus and mean single locus outcrossing rates

(tm,

ts)

indicated that about 15

%

selfing can occur in the studied populations. The heterogeneity between pollen and ovule allele

frequencies was low suggesting population structuration, in agreement with the estimates of correlation of

tm

within

progeny

(rt)

and correlation

of

outcrossed paternity

(rp).

The difference of

FIs

estimates between offspring and mother

plants suggest some selection favouring heterozygotes between seedling and adult stages.

Cecilia Bessega, Departamento de Ciencias Biologicas, Facultad Ciencias Exactas

y

Naturales, Universidad de Buenos Aires,

1428

Buenos

Aires, Argentina.

E-mail: cecib@bg.fcen.uba.ar

Some species

of

the section Algarobia of genus

Prosopis

constitute important natural resources in

arid and semiarid regions of the world. They are

promising multipurpose species for reforestation, pro-

duction of wood, charcoal, forage, and human food.

Their breeding and improvement have been recom-

mended by several authors

(BURKART

1976;

FELKER

1979;

LEAKEY

and

LAST

1980;

GALINDO-ALMANZA

1983;

GALINDO-ALMANZA

and

GARCIA

1986a,b). To

reach this goal it is paramount to know the biology,

genetic variability and differentiation, mating system,

genetic structure, and adaptive strategies of these

species.

The species

so

far studied

of

this section exhibit

high variability in morphological characters such as

production, size and shape

of

fruits, life form, growth

rate, adaptability to extreme temperature and salinity

(FELKER

1979;

GALINDO-ALMANZA

1983;

ROIG

1993). Important intraspecific genetic variability was

also detected by means

of

isoenzyme electrophoresis

(SAIDMAN

1985, 1986, 1988, 1990, 1993;

SAIDMAN

and

VILARDI

1987, 1993;

SAIDMAN

et al. 1997,

*

Fellow of Consejo Nacional de Investigaciones Cientificas

y

t

Fac. Ciencias Exactas, Fisicas

y

Naturales. Universidad de

Member of the Consejo Nacional de Investigaciones Cienti-

Tecnicas, (CONICET).

Cordoba. Argentina.

ficas y Ttcnicas. Argentina.

1998a) and random amplified polymorphic

DNA

seg-

ments

(SAIDMAN

et al. 1998b; Bessega submitted).

There are strong evidence of frequent hybridization

among several species, coming from morphological,

biochemical and isoenzymatic studies

(PALACIOS

and

BRAVO

1981;

NARANJO

et al. 1984;

HUNZIKER

et al.

1986;

SAIDMAN

1990;

VERGA

1995). The virtual lack

of clear reproductive barriers as well as the high

genetic similarity between “good taxonomic species”

has led to the assumption that several sympatric

species of this section inhabiting the Chaqueiia Bio-

geographical Region in Argentina would constitute a

syngameon

(PALACIOS

and

BRAVO

1981;

SAIDMAN

1985;

GRANT

1989).

Both facts, high intraspecific variability and inter-

specific hybridization rate have been related with

floral biology.

For

a long time these species were

assumed to be protogynous

(BURKART

1937, 1952,

1976) and this property together with lack

of

clear

reproductive barriers was considered as responsible

of an outcrossing mating system which, in time,

might explain the high intraspecific variability and

the occurrence of interspecific hybridization

(SOL-

BRIG

and

BAWA

1975;

SOLBRIG

and

CANTINO

1975;

NEFF

et al. 1977;

SIMPSON,

1977;

SIMPSON

et al.

1977;

HUNZIKER

et al. 1986). However, according to

more recent results this assumption should be revised.

Studies of maturation

of

flowers and nectar produc-

20

C.

Bessega et al.

Hereditas

132 (2000)

tion in three Southamerican species of this section

(P.

flexuosa,

P.

chilensis

and

P.

pugionata)

made by

GENISSE et al. (1990) do not support this point. They

concluded that flowers are not protogynous and sug-

gested that the high variability in some species of

Algarobia would be maintained by an autoincompat-

ibility system independent

of

protogyny

.

Besides,

GALINDO-ALMANZA et al. (1992) observed through

pollination studies that Mexican populations of

P.

glandulosa

var.

torreyana

and

P.

iaevigata

are at least

partially autocompatible.

KEYS

and SMITH (1994)

studying the mating system in three populations of

P.

velutina

from

U.S.A.

(using isoenzymatic markers)

concluded that the outcrossing rate is about 90

YO

and that selfing can occur at a low rate. Finally,

studies of population structure based on isozyme

electrophoresis using WRIGHT'S (195 1)

F

statistics

(KEYS

and SMITH 1994; SAIDMAN et al. 1998a) show

a general trend to homozygote excess in seedlings

collected from natural populations, which might be

compatible with a certain degree of selfing. However,

Prosopis

species populations are expected to be struc-

tured because pollen and seed dispersals are limited.

The endozoic seed dispersal determines that seeds

from the same mother plant are eaten by herbivores

and transported away jointly. They are then de-

posited in dung and full or half

sib

seeds tend to

germinate together in a narrow area. Besides, the

pollination

is

entomophilous (GENISSE et al. 1990)

favouring crosses among near neighbour plants.

Therefore, the homozygote excess might also be con-

sequence of population substructure.

Taking into account the contradictory data avail-

able on the mating system of these species our objec-

tives were to estimate mating system and population

structure parameters

of

seven species of the section

Algarobia of genus

Prosopis.

During the analysis of

our data we also tested the first genetic interpretation

(based on indirect evidence) of the isozyme bands

described in previous works (SAIDMAN 1985, 1986,

1988, 1990, 1993; SAIDMAN and

VILARDI

1987, 1993;

SAIDMAN et al. 1997, 1998a) by analyzing family

data.

Table

1.

Geographic location

of

population analyzed

MATERIALS

AND

METHODS

Sampling description

The sampling involved one population of each of

seven species of section Algarobia that occur in semi-

arid regions of the Chaqueiia Biogeographic Region

in Argentina, and Texas and Arizona in USA (Table

1).

Each population

is

a fairly continuous forest

of

several square kilometres wide. The populations

sam-

pled are isolated from each other and apparently are

made up from pure stands, with no evidence

of

interspecific hybridization. Six of the studied species,

P.

glandulosa,

P.

velutinu,

P.

chilensis,

P.

nigra,

P.

alba,

and

P. flexuosa

belong to the series Chilenses,

and the remaining one,

P.

ruscifolia,

to the series

Ruscifoliae.

At

least 10 mother plants were sampled in each

population from Argentina. The samples from popu-

lations from United States were kindly donated by

Dr.

C.

J.

De Loach (Grassland Research Station-

USDA/ARS). They included

5

mother plants. In all

cases the trees were separated at least

50

m. from

each other. This distance between sampled mothers

plants reduce the probability that they interbreed,

because enthomophilic pollination is related with lim-

ited pollen dispersal. About

50

pods were collected

from each mother plant. All seeds from each tree

were stored in single bags. From each of these bags

seeds were randomly sampled for the isozymal analy-

sis

of each progeny array. The number of seeds

analyzed per mother plant (family array size) was

about 10 in Argentinean species and 15 in species for

USA.

Genetic characterization

A total of 7 isoenzymatic system which were shown

to reveal polymorphic and codominant loci (SAID-

MAN

1985, 1986,

1988,

1990, 1993; SADMAN and

VILARDI 1987, 1993; SAIDMAN et al. 1997, 1998a)

were used

in

the current study: alcohol dehydroge-

nase (ADH), esterase (EST), glutamate oxalacetate

transaminase (GOT), amino peptidase (AMP), 6-

phosphogluconate dehydrogenase (6PGD), isocitrate

Serie Species Population Latitude Longitude

Chilenses

P.

alba

Burruyacu 26'30 64"43'

P.

nigra

Huilla Catina 27'32' 64"06'

P.

chilensis

Patquia

30'02'

66"52'

P.

flexuosa

Quilmes 26"22

65'58'

P.

velutina

Santa Rita

31'35'

110"53'

Ruscifoliae

P.

ruscifolia

Rivadavia 24'1

1'

62"53'

P.

glandulosu

Weslaco 26'09' 9799'

Hereditas

132

(2000)

Mating system in species

of

Prosopis (Leguminosae)

21

Table

2.

Enzyme structure

of

the analyzed systems

Enzyme

system

Gene symbol EC Structure

No

of

loci de- Max.

No

of alleles detected

tected

Alcohol dehydrogenase

Adh

1.1.1.1

Dimeric

Aminopeptidase

Amp

3.4.1

1.1

Monomeric

Esterase

Est

3.1.1

Monomeric

Glutamate dehydrogenase

Got

2.6.1.1

Dimeric

Isocitrate dehydrogenase

Zdh

1.1.1.42

Dimeric

6-phosppgluconic

dehydro-

6-

Pgd

1.1.1.43

Dimeric

Shikimic

dehydrogenase

Skd

1.1.1.25 Monomeric

genase

dehydrogenase (IDH) and shikimic dehydrogenase

(SKD). The technique employed was horizontal elec-

trophoresis on polyacrylamide gels. The methods em-

ployed for the former five systems are described in

SAIDMAN (1985, 1986). For IDH and

SKD

the

method was modified from VERGA (1995). The ho-

mogenates for ADH were made from 24 h soaked

seeds; for the remaining systems 5-7 day old cotyle-

dons were used.

Controlled crosses in

Prosopis

species have not

been successful

so

far.

In cases like this one, the

genetic interpretation of the isozyme data is usually

based on

(1)

the presence of banding patterns typical

for specific isozymes;

(2)

biochemical and develop-

mental allelism tests (SAIDMAN 1985, 1986);

(3)

the

possibility of a maternal genotype that could have

generate the progeny array; and

(4)

the fit of the

progeny array data to the expected Mendelian segre-

gation ratios for a diploid organism (KEYS and

SMITH 1994; VERGA 1995). In species of the section

Algarobia additional support to the genetic interpre-

tation was obtained from the analysis of segregating

patterns of natural hybrids (SAIDMAN 1985, 1986,

1990). In the present study the analysis of segregation

in progeny arrays was used to support previous hy-

potheses on the genetic determination of isozyme

bands.

Loci were numbered consecutively, with the most

anodal being designated locus

1.

Alleles within loci

were designated by a number referring to their rela-

tive mobility on the gel respect to bromophenol.

Data analysis

Genetic structuration

Allelic and genotypic frequencies

of

the whole seed

population sample was estimated including all avail-

able loci. Similar numbers of seeds from different

mother plants were analyzed. The bias from Hardy-

Weinberg expectations were evaluated by means of

the

F,s

fixation index (WRIGHT 1951) estimated by

the method of NEI (1977) using the program Biosys

1.7 (SWOFFORD and SELANDER 1981).

Mating system parameters

Estimates of multilocus

(t,)

and mean single locus

(t,)

outcrossing rates, correlation of

tm

within

progeny arrays

(rt),

the correlation of outcrossed

paternity

(rp),

and fixation index of maternal parents

(F,sM)

were calculated using the MLTR program (K.

Ritland, Department of Forest Science, University

of

British Columbia), an improved version of the MLT

(RITLAND 1990a) computer program. This program

is based on the multilocus mixed-mating model and

the estimation procedure of RITLAND and JAIN

(1 98 1) which assumes that progeny are derived from

either random mating (outcrossing) or self-fertiliza-

tion. The maternal genotypes for each family is in-

ferred by the method of BROWN and ALLARD (1 970).

The estimation of mating system parameters was

made by the Expectation-Maximization method to

assure convergence.

The analysis of mating system parameters was

limited to those loci which could be simultaneously

studied in each individual. The mixed-mating model

assumes independent segregation of alleles at the

different marker loci. In order to support this as-

sumption with the available data we tested the null

hypothesis of no association between genotypes at

different loci. The absence of such associations can be

considered as evidence for independent segregation

(see

DOLIGUEZ

and JOLY 1997). Possible genotypic

associations among these loci were tested using the

program GENEPOP (RAYMOND and ROUSSET

1995). The method creates contingency tables for all

pairs of loci in each population and then performs a

probability test (or Fisher exact test) for each table

using a Markov chain.

Estimated pollen and ovule allele frequencies were

compared by means

of

a contingency table using the

Pearson Chi-square test.

22

C.

Bessega et

al.

Hereditas

132

(2000)

RESULTS

Genetic interpretation

The analysis

of

segregation within progeny arrays

allowed to confirm previous

(SAIDMAN

1985,

1986,

1990)

interpretation of the genetic determination

of

isozyme bands. In all families the maternal genotype

could be inferred from progeny genotypes. The sub-

unit structure (monomeric or dimeric) could be deter-

mined on the basis of the number of bands observed

in heterozygous individuals. Table

2

summarizes the

isozyme structure and the number

of

alleles detected

for

each locus.

Genetic structure

Allelic frequencies and fixation indices for the whole

seed (offspring) sample are listed in Table

3.

In all

cases

Frs

in this sample

(Frso)

was positive, varying

Table

3.

Allelic frequencies and fixation index

(Frso)

estimated in the offspring populations.

N:

sample size,

SE:

standard error

of

FIso

P.

alb

P.

nig

P.

chi

P.

j7e

P.

rus

P.

vel

P.

gla

Adh-1

Adh-2

Amp-2

Est

-

1

Got-1

Got-2

Zdh

-

1

Zdh-2

6-Pgd-

1

6-

Pgd-2

Skd-

1

N

SE

FISO

130

124

217

214

276

270

193

1

92

191

190

172

171

170

1

69

254

248

240

234

227

170

1

63

230

130

1

220

288

16'

1100

1

243

226

126

10

22

I

20

1

22

223

1

24

119

0.000

1

.ooo

0.000

1.000

0.000

0.406

0.594

0.000

0.889

0.111

0.000

0.012

0.000

0.988

0.000

0.034

0.966

0.000

0.093

0.886

0.021

0.000

1.000

0.000

1

.ooo

0.000

0.230

0.098

0.672

0.144

0.561

0.295

0.389

0.107

90

0.579

0.412

0.009

0.000

1

.ooo

0.000

0.127

0.461

0.412

0.289

0.579

0.132

0.000

0.006

0.000

0.570

0.424

0.000

0.280

0.699

0.021

0.000

0.013

0.176

0.782

0.029

0.000

1.000

0.43 1

0.569

0,000

0,067

0.008

0.925

0.353

0.549

0.098

0.242

0.161

100

0.120

0.880

0.000

1

.ooo

0.000

0.256

0.565

0.179

0.000

0.125

0.634

0.241

0.163

0.000

0.837

0.000

0.134

0.866

0.000

0.258

0.677

0.065

0.000

0.632

0.368

0.531

0.250

0.219

0.625

0.062

0.313

0.750

0.03 1

0.219

85

0.175

0.086

0.490

0.388

0.122

0.000

1

.ooo

0.000

0.455

0.545

0.000

0.833

0.161

0.006

0.516

0.000

0.437

0.047

0.000

0.314

0.637

0.049

0.000

0.005

0.116

0.858

0.021

1

.ooo

0.000

0.0

10

0.990

0.000

0.118

0.198

0.684

0.587

0.207

0.206

0.391

0.139

90

1.000

0.000

0.058

0.926

0.016

0.387

0.41

1

0.202

0.000

0.886

0.114

0.000

0.051

0.000

0.949

0.000

0.591

0.409

0.000

0.391

0.609

0.000

1

.ooo

0.628

0.372

0.000

0.256

0.161

0.583

0.076

0.924

0.000

0.295

0.085

84

0.780

0.220

0.000

1.000

0.000

0.794

0.206

0.000

0.097

0.742

0.161

0.116

0.802

0.000

0.08

1

0.000

1.000

0.000

0.250

0.750

0.000

1.000

0.000

1.000

0.000

1

.ooo

0.531

0.469

0.000

0.312

0.175

74

0.936

0.064

0.000

1

.ooo

0.000

0.417

0.466

0.117

0.000

0.094

0.469

0.438

0.077

0.055

0.440

0.363

0.066

0.000

0.608

0.315

0.000

0.077

0.054

0.920

0.026

0.000

1

.ooo

0.000

1.000

0.000

1

.ooo

0.093

0.651

0.256

0.383

0.103

91

Hereditas

132

(2000)

Mating system in species of Prosopis (Leguminosae)

23

Table

4.

SigniJcance levels for chi-square tests of independence of genotypes between loci (Fisher exact test).

NS:

p

>

0.05;

*:

p

<

0.05;

**:

p<

0.01.

In no case the association was sign8cant

at

matrix level

Amp-1

Est-1

Got-1

Got-2

6Pgd-1 6Pgd-2 Idh

-

1

Idh -2

Est

-

1

Got-1

Got-2

6Pgd-

1

6Pgd-2

Idh

-

1

Idh -2

Skd-

1

NS

-

NS

**

NS

*

NS

-

NS

*

NS

NS NS

NS

*

from 0.175 to 0.391, indicating a general trend to-

wards homozygote excess within populations.

Mating system parameters

Since the mixed mating model assumes independence

among loci, possible genotypic associations were

tested within each species and across species. The

trends observed were similar in all populations and

the results are summarised in Table 4. Only 6 of a

total of 31 locus pairs showed significant association,

but in no case the disequilibrium was significant at

matrix level applying the Bonferroni sequential test.

Since genotypes for most pairs of loci were indepen-

dent the bias in the results due to physical linkage

between loci were considered negligible.

Ovule and pollen allele frequencies (Table

5)

were

compared through contingency table analysis. Out of

44 comparisons only 8 yielded significant differences.

These result suggests low heterogeneity between pol-

len and ovule frequencies.

Multilocus

(tm)

and average single locus

(ts)

out-

crossing rates were similar to each other (Student

test:

T6

=

0.98,

P

=

0.36). Multilocus outcrossing rate

estimates in the studied species varied from 0.718 in

P.

alba

to

1

in

P. nigra

(Table

6),

indicating that these

species are mostly outcrossing but selfing can occur at

least in some populations. The correlation of

tm

within progeny arrays

(rt)

in most cases was high,

suggesting that outcrossing rates differ among mother

plants. The correlation of outcrossed paternity

(rp)

was also high, indicating that many individuals

within a progeny array are full sibs.

The estimates of fixation indices for maternal geno-

types

(FlsM)

were in almost all cases lower than the

corresponding values for their offspring’s

(Ffs,)

(see

Tables 3 and

5),

and in some species the confidence

interval of

FIsM

includes zero. The comparison of

Frs

between maternal and offspring genotypes yielded

highly significant differences (Student test:

T6

=

7.87,

P

=

0.0002).

DISCUSSION

Classical studies in species of the Section Algarobia

(SIMPSON 1977) pointed that these species are protog-

ynous and obligate outcrosser. This view contrasts

with more recent studies of floral biology by

GENISSE

et al. (1990) that indicate that species of Algarobia

are not protogynous. However, this authors were yet

prone to accept that these species are obligate out-

crosser and hypothesise that this condition would be

consequence of some autoincompatibility system.

However, results of isozyme analysis showed sig-

nificant excess of homozygotes in all populations

so

far studied of species of this section (SAIDMAN 1985,

1986, 1988, 1990, 1993; SAIDMAN and VILARDI 1987,

1993; SAIDMAN et al. 1997, 1998a, VERGA 1995,

KEYS and SMITH, 1994), which suggests high levels of

inbreeding. Such inbreeding would be expected if

there is family structure within populations, but it

might also be caused by some degree of selfing.

The last possibility seem to be supported by differ-

ent pollination studies showing that

P.

glandulosa,

P.

laevigata

(GALINDO-ALMANZA et al. 1992) and

P.

velutina

(KEYS 1993; KEYS and SMITH 1994) are at

least partially selfcompatible. Solving the question

about the relation between mating system and ho-

mozygote excess in species of section Algarobia re-

quires testing the genetic interpretation of isozyme

bands, and analysing mating system parameters and

population structure.

In the current study the genetics of the loci were

inferred from single-tree progeny arrays. The number

of loci considered to control the isozyme systems

studied here were similar to the results of previous

studies (SOLBRIG and BAWA 1975; SAIDMAN 1985,

1986, 1988, 1990, 1993; SAIDMAN and VILARDI 1987,

1993; SAIDMAN et al. 1997, 1998a;

VERGA

1995). In

agreement with other studies in species of Algarobia

all populations analyzed here showed high ho-

mozygote excess for the seed population

(F,,,

>

0).

Mating system parameters in species of genus Prosopis (Leguminosae).

Summary (2 min read)

Some species of the section Algarobia of genus

Sampling description

Genetic characterization

Genetic structuration